Method of identifying agents that inhibit quorum sensing activity of gamma-proteobacteria

ABSTRACT

Screening assays that allow for the identification of agents that increase acyl homoserine lactone (AHL) acylase expression and/or AHL acylase activity in γ-proteobacteria such as  Pseudomonas aeruginosa . Such agents are useful, for example, for inhibiting quorum sensing activity of such bacteria by increasing degradation of long chain, but not short chain, AHLs and, therefore, can be useful for treating infections by such bacteria.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Application Ser.No. 10/861,224 filed Jun. 3, 2004, now U.S. Pat. No. 7,335,352; whichclaims the benefit under 35 USC §119(e) to U.S. Application Ser. No.60/574,366 filed May 25, 2004, now abandoned and to U.S. ApplicationSer. No. 60/475,745 filed Jun. 4, 2003, now abandoned. The disclosure ofeach of the prior applications is considered part of and is incorporatedby reference in the disclosure of this application.

GRANT INFORMATION

This invention was made with government support under Grant No.DBI-0107908 awarded by the National Science Foundation. The UnitedStates government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to screening assays, and morespecifically to methods of identifying agents that can modulate theexpression and/or activity of an acyl homoserine lactone (AHL) acylasethat breaks down long chain AHLs, but not short chain AHLs, inγ-proteobacteria such as Pseudomonas aeruginosa, to agents identified bysuch methods, and to methods of using the agents to treat aγ-proteobacteria infection by inhibiting quorum sensing activity by thebacteria.

2. Background Information

Pseudomonas aeruginosa, an opportunistic pathogen, is a gram-negativeγ-proteobacteria. P. aeruginosa can thrive in a variety of environments,requiring only minimal nutrients and moisture. For example, P.aeruginosa exists in soil, water, and on animals. P. aeruginosa releasesenzymes such as an elastase, an alkaline protease, and a cytotoxin thataid in the invasion and destruction of host tissues. In an infectedhost, P. aeruginosa often invades small arteries and veins, whichfrequently leads to metastatic nodular lesions in the lungs. Pseudomonasinfections can be aggressive, often result in sepsis, and are associatedwith a high mortality rate.

Immunocompromised individuals such as patients with burns, urinary tractinfections, or cystic fibrosis are particularly susceptible toPseudomonas infections. In cystic fibrosis patients, for example,pneumonia due to P. aeruginosa infection is common, likely due toaccumulated bronchial secretions providing an environment in which thePseudomonas can flourish. Further, P. aeruginosa is extremely resistantto antibiotics and, therefore, treatment must be aggressive and,nevertheless, is often unsuccessful. Thus, a need exists for drugs thatcan be used to successfully treat Pseudomonas infections. The presentinvention satisfies this need, and provides additional advantages.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery thatγ-proteobacteria such as Pseudomonas aeruginosa express an acylhomoserine lactone (AHL) acylase that can degrade long chain, but notshort chain, AHLs to produce fatty acid and homoserine lactone (HSL),and that such AHL acylase activities can regulate quorum sensingactivity in the γ-proteobacteria. As such, the invention providesmethods to identify agents that can modulate γ-proteobacterium AHLacylase expression and/or activity, including high throughput screeningmethods, and further provides a means to identify agents that are usefulfor treating patients having, for example, a Pseudomonas infection,including agents that are useful for a particular patient, thus allowingfor personalized medicine.

Accordingly, the present invention relates to a method of identifying anagent that modulates γ-proteobacterium long chain AHL acylase expressionand AHL acylase activity. Such a method can be performed, for example,by contacting at least one sample (e.g., 1, 2, 3, 4, 5, etc.), whichcontains (or to which is/are added) the AHL acylase and a long chainAHL, with a test agent, under conditions suitable for AHL acylaseactivity, and detecting a change in AHL acylase activity in the presenceof the test agent as compared to the AHL acylase activity in the absenceof the test agent, wherein a change in AHL acylase activity identifiesthe test agent as an agent that modulates the γ-proteobacterium longchain AHL acylase activity. A method of the invention can be used toidentify an agent that increases AHL acylase activity or an agent thatdecreases AHL acylase activity, as well as an agent that increasesexpression of an AHL acylase gene in a γ-proteobacterium (e.g., PvdQgene expression in P. aeruginosa), thereby increasing AHL acylase levelsand AHL acylase activity, or an agent that decreases AHL gene expressionin a γ-proteobacterium.

The long chain AHL for which AHL acylase activity is examined can be anylong chain AHL that can be degraded by the AHL acylase to a fatty acidand HSL, including, for example, N-3-octanoyl-DL-homoserine lactone(C8HSL), N-3-decanoyl-DL-homoserine lactone (C10HSL),N-3-dodecanoyl-DL-homoserine lactone (C12HSL),N-3-oxododecanoyl-L-homoserine lactone (3OC12HSL), orN-3-tetradecanoyl-DL-homoserine lactone (C14HSL). The γ-proteobacteriumthat contains the AHL acylase, or from which the AHL acylase is derived,can be any γ-proteobacterium of interest, including, for example,medically important γ-proteobacterium such as a Pseudomonas species(e.g., P. aeruginosa), a Vibrio species (e.g., F. cholerae), or anAzotobacter species. For example, the AHL acylase can be Pseudomonasaeruginosa PvdQ AHL acylase, which has an amino acid sequence as setforth in SEQ ID NO:2, or a Pseudomonas PA1032 AHL acylase, which has anamino acid sequence as set forth in SEQ ID NO:4 or SEQ ID NO:5.

In one aspect of a method of the invention, the sample can furthercontain (or have added thereto) a short chain AHL. As disclosed herein,the γ-proteobacterium AHL acylase lacks the ability to metabolize(degrade) short chain AHLs. As such, this aspect of a method of theinvention provides a means to confirm that a test agent that, forexample, increases AHL acylase activity with respect to long chain AHLbreakdown, has no effect on the short chain AHL (i.e., by detecting nochange in the amount of the short chain AHL in the presence of the testagent as compared to the absence of the test agent). A short chain AHLuseful in this aspect can be any short chain AHL that is not normallydegraded by the AHL acylase being examined, including, for example,N-3-butanoyl-DL-homoserine lactone (C4HSL), N-3-hexanoyl-L-homoserinelactone (C6HSL), N-3-oxohexanoyl-L-homoserine lactone (3OC6HSL), orN-3-heptanoyl-DL-homoserine lactone (C7HSL).

A sample examined according to a method of the invention can be anysample that contains (or to which can be added) an AHL acylase, suchthat conditions are suitable for AHL acylase activity. Such conditions,which can include, for example, an appropriate concentration of ironions, are selected based on whether the assay is performed in a cellfree format (e.g., using purified reactants such as purified AHL acylaseand/or purified long chain AHLs, etc.) or is performed using a cellbased assay. Accordingly, in one embodiment, the method is performed invitro, wherein the AHL acylase comprises purified AHL acylase, which canbe obtained, for example, from an extract comprising a γ-proteobacteriumthat expresses the AHL acylase (e.g., an extract comprising aPseudomonas species such as P. aeruginosa), or from an in vitrotranslation or coupled transcription/translation reaction using apolynucleotide encoding the AHL acylase (e.g., a polynucleotide as setforth in SEQ ID NO: 1 or in SEQ ID NO:3).

In another embodiment, the method is performed as a cell based assay,wherein the sample comprises a cell sample, or an extract of a cell, andwherein the AHL acylase is expressed in the cell. The cell can be aγ-proteobacterium, in which the AHL acylase is expressed in nature(e.g., a Vibrio species such as V. cholerae), can be a host cell ortissue sample that is infected with γ-proteobacteria (e.g., a biopsysample from a subject infected with the bacteria), or can be a cell thathas been genetically modified to express a polynucleotide encoding aγ-proteobacterium AHL acylase (e.g., a host cell transformed,transfected or transduced with a polynucleotide encoding a PseudomonasPA1032 AHL acylase as set forth in SEQ ID NO:4 or SEQ ID NO:5). In oneaspect of this embodiment, the sample comprises a cell, tissue orbiologic fluid obtained from a subject having a γ-proteobacteriuminfection. In another aspect, the sample comprises a cell, tissue orbiologic fluid obtained from a subject having a Pseudomonas infection.The subject can be any subject susceptible to infection by theγ-proteobacterium, including any vertebrate such as a mammal (e.g., ahuman subject infected by P. aeruginosa or V. cholerae).

According to the present methods, a change in AHL acylase activity canbe detected using any assay suitable for measuring AHL acylase activity,including, for example, assays that can measure AHL levels and or AHLbreakdown products such as a fatty acid and/or homoserine lactone (HSL)in the sample (or an aliquot or fraction of the sample). Such methodscan be based on the chemical structure of the substrate (long chain AHL)and/or product (e.g., HSL) of the AHL acylase, including, for example,mass spectroscopy, which can measure the amount of AHL and/or HSL in thesample, and thin layer chromatography, which can measure the amount ofHSL in the sample. AHL acylase activity can also be measured using afunctional (e.g., biological) assay, including, for example, contactingthe sample or an aliquot or fraction of the sample with aγ-proteobacterium (e.g., a Pseudomonas species) and detecting the amountof quorum sensing activity, or can be measured using an availablebioassay strain that can be used to detect and determine theconcentration of AHLs.

In one embodiment, a method of the invention is performed in a highthroughput format, thus allowing for the screening, in parallel, of oneor more test agents with one or more samples, wherein the agents and/orsamples independently are the same or different. As such, the methodallows for testing one or more concentrations of one or more test agentsto identify a concentration of an agent particularly useful formodulating AHL acylase activity. Further, the method allows forexamining several same test agents on one or a plurality of samesamples, and/or one or more different test agents on several samesamples, thus providing a means to obtain statistically significantresults. Also, the method allows for examining one or a plurality ofcell sample(s) taken from a subject having a γ-proteobacterium (e.g., P.aeruginosa) infection with one or a plurality of the same (e.g.,different concentrations) or different test agents, to identify an agentthat is best suited, for example, for increasing AHL acylase activity inthe patient, thus increasing the rate of breakdown of long chain AHLsproduced by the γ-proteobacterium. Such an agent can be useful forreducing or inhibiting quorum sensing activity by the infectingbacteria, thereby ameliorating the infection in the subject.

The screening assays of the invention, particularly when utilized in ahigh throughput format, provide a means to screen one or more librariesof test agents, including, for example, a combinatorial library of testagents, which can be a random library, a biased library, or a variegatedlibrary of test agents. For example, the method can be used to screen acombinatorial library of randomly generated test agents, then, ifdesired, positive agents that desirably modulate AHL acylase activity(e.g., increase AHL acylase activity) can be used to generate a libraryof biased and/or a variegated test agents based on the structure of theidentified positive agent(s) to obtain an agent that modulates the AHLacylase activity and, for example, has additional desirable propertiessuch as enhanced stability in a biological system (e.g., a subject to betreated with the agent), useful absorption characteristics, or the like.

The present invention also relates to an agent identified by a method ofthe invention. Such an agent can be, for example, a peptide, apolynucleotide, a small organic molecule, or a peptidomimetic. Where theagent is to be used for a therapeutic method, it can be formulated in aform suitable for administration to a subject, for example, as a pill ora liquid, and can be administered, for example, orally, by injection, orvia inhalation. Accordingly, methods are provided for treating a subjectinfected with a γ-proteobacterium (e.g., P. aeruginosa) by administeringan agent identified by a screening assay of the invention to thesubject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts two mechanisms by which AHLs can be inactivated.Mechanism A represents cleavage of the amide bond by bacterial AHLacylase, which yields HSL and the corresponding fatty acid (21, 23). TheAHL amide bond is chemically stable under conditions of nonextremetemperature and pH. Mechanism B represents cleavage of the lactone ringby bacterial AHL lactonase, yielding the corresponding acyl-homoserine(7, 46). The lactone ring is also subject to chemical hydrolysis; thechemical half-life of the ring is about 10⁽7−pH) days, i.e., it is lessstable with increased alkalinity. The acyl side chain diversity ofknown, naturally occurring AHLs has been reviewed (11).

FIG. 2 shows rRNA-based phylogeny of strain PAI-A. Construction of thephylogram used 1,120 unambiguously aligned nucleotide positions in a10,000-step Tree Puzzle 5.0 maximum-likelihood analysis (36, 39; seeExample 1 for GenBank Ace. Nos.). The bar represents evolutionarydistance as 0.01 changes per nucleotide position, determined bymeasuring the lengths of the horizontal lines connecting the species.The numbers provide support for the robustness of the adjacent nodes.The arrow points to the short node from which the five strains withinthe shaded box radiate.

FIGS. 3A to 3D show LC/APCI-MS analysis of a cell-free fluid sampledfrom a P. aeruginosa culture utilizing C10HSL as a sole energy source inMES 5.5 medium.

FIG. 3A shows a chromatogram showing the separation of homoserine and/orHSL, MES buffer, and decanoyl-HSL (left axis). The hatch markscorrespond to changes in methanol/water solvent ratios during the courseof the run (right axis).

FIG. 3B shows the mass spectrum of the first peak, which resolveshomoserine from homoserine lactone. The peak tail can overlap with, butcan be resolved from, the component in the second peak.

FIG. 3C shows the mass spectrum of the second peak, morpholinoethanesulfonic acid (MES buffer).

FIG. 3D shows the mass spectrum of the third peak, decanoyl-HSL. Thismethod can be applied to separate and determine the concentrations of anumber of other AHLs and any acyl-homoserine degradation products.

FIG. 4 shows growth of P. aeruginosa PAO1 in ammonia-replete MES 5.5media containing 1 mM 3OC12HSL as the sole energy source. Substrateconsumption and product accumulation were determined via LC/APCI-MS.Note that since the 3OC12HSL substrate was poorly soluble at the initialconcentrations employed; virtually no AHL was observed in the culturefluid at the time of inoculation. As growth progressed, a transientspike of AHL in solution was observed. HSL accumulated throughout thegrowth phase but was degraded upon entry into stationary phase yieldinga transient intermediate, homoserine. 3OC12-homoserine concentrationsremained static throughout the course of the experiment and neverexceeded 0.1% of the initial AHL concentration. Culture pH was wellcontrolled throughout the study.

FIG. 5 shows activity of E. coli cells expressing recombinant PvdQ (SEQID NO:2), which degraded 3OC12HSL and generated stoichiometric amountsof HSL. Substrate disappearance and product accumulation were determinedby LC/APCI-MS. Induced cells containing the pPvdQ-PROTet plasmid werewashed and suspended in MOPS (pH 7.2) buffered medium to a final OD₆₀₀of 1.2. AHL degradation and HSL accumulation were not observed over theduration of the experiment in either heat-killed suspensions of the samecells, or in no-cell controls.

FIG. 6 shows growth and accumulation of endogenous 3OC12HSL by P.aeruginosa PAO1 wild type (▴, Δ), and a recombinant derivativeconstitutively expressing PvdQ (▪, □). Because of the organiccomplexities of LB, sampled cell-free culture fluids were extracted withethyl acetate before LC/APCI-MS analysis; the limit of detection for3OC12HSL was 75 nM and was plotted in place of zero. Cultures were grownat 30° C. in LB. Under similar culture conditions, a PvdQ knockoutmutant grew and accumulated 3OC12HSL in parallel with the wild type.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the discovery thatγ-proteobacteria such as Pseudomonas aeruginosa (P. aeruginosa) expressan AHL acylase that can degrade long chain, but not short chain, acylhomoserine lactones (AHLs) to produce fatty acid and homoserine lactone(HSL), and that such AHL acylase activity regulates quorum sensingactivity in γ-proteobacteria. Acyl-homoserine lactones (AHLs) areemployed by several Proteobacteria as quorum sensing signals. Paststudies established that these compounds are subject to biochemicaldecay and can be used as growth nutrients. As disclosed herein, a soilbacterium, Pseudomonas strain PAI-A, was isolated. Pseudomonas strainPAI-A degraded N-3-oxododecanoyl-L-homoserine lactone (3OC12HSL) andother long chain acyl, but not short chain acyl AHLs as sole energysources for growth.

The small sub-unit rRNA gene from strain PAI-A was 98.4% identical tothat of P. aeruginosa, but the soil isolate did not produce obviouspigments or AHLs or grow under denitrifying conditions or at 42° C. Thequorum sensing bacterium P. aeruginosa, which produces both 3OC12HSL andC4HSL, was examined for the ability to utilize AHLs for growth, and likePAI-A, used AHLs for growth, showing a similar specificity for thedegradation of long acyl, but not short acyl AHLs. In contrast to strainPAI-A, P. aeruginosa PAO1 growth on AHLs commenced only after a long lagphase. Liquid chromatography-atmospheric pressure chemicalionization-mass spectrometry (LC/APCI-MS) analyses indicated that strainPAO1 degraded long acyl AHLs via an AHL acylase and ahomoserine-generating HSL lactonase (see Example 1). A P. aeruginosagene, PvdQ (PA2385), was previously identified as a homologue of the AHLacylase described from a Ralstonia species. As disclosed herein, E. coliexpressing PvdQ catalyzed the rapid inactivation of long acyl AHLs andthe release of HSL (see Example 2). P. aeruginosa engineered toconstitutively express PvdQ did not accumulate its 3OC12HSL quorumsignal when grown in rich media. However, PvdQ knockout mutants of P.aeruginosa utilized 3OC12HSL at wild-type growth rates and yields.

The present results demonstrate that pseudomonads or otherγ-proteobacteria degrade AHLs, that quorum sensing bacterium have AHLacylase activity, that bacteria have HSL lactonase activity, and thatAHL degradation by γ-proteobacteria is specific for AHLs with long sidechains. Accordingly, the present invention provides methods to identifyagents that modulate γ-proteobacteria long chain AHL acylase activity.In addition, the present invention provides compositions containingagents identified by such a method.

Many bacterial species control and modulate their physiology in responseto increases in their population densities in a process known as quorumsensing (12, 25). Several dozen species of Proteobacteria use AHLs asdedicated signal molecules in this process. A diversity of acyl-HSL,structures and the enzymes and proteins involved in their synthesis andrecognition have been elucidated (13, 27, 31, 35). One of the beststudied quorum sensing species is the opportunistic pathogen Pseudomonasaeruginosa, which makes and responds to two distinct acyl-homoserinelactones: N-3-oxododecanoyl-L-homoserine lactone (3OC12HSL; also knownas PAI, the Pseudomonas autoinducer of the las QS system), andN-3-butanoyl-DL-homoserine lactone (C4HSL; also known as PAI-2, theautoinducer of the rhl QS system). The two quorum circuits controlseveral physiologies and virulence factors associated with the infectionof immunocompromised individuals, such as those with cystic fibrosis(40). The influence of AHLs on the global regulation of gene expressionby P. aeruginosa is vast (37, 42). AHL-mediated signaling and signaldynamics are very important to the biology of this species, and,therefore, it is important to understand issues relating to signalstability so that methods can be developed for modulating quorum sensingactivity.

Quorum sensing activity provides a means of cell-to-cell communication(“cell-to-cell signaling”). A “quorum” generally comprises a densepopulation of cells having the capability to communicate with eachother. A quorum of bacterial cells have the ability to “sense” theirdensity level and, as a result, can act as a group of cells rather thanas individual cells (see, e.g., Fuqua and Greenberg, Molecular CellBiology Vol. 3: 685-695, Nature Reviews 2002; Van Delden and Iglewski,Emerging Infectious Diseases Vol. 4, No. 4: 1-10, 1998; each of which isincorporated herein by reference). Degradation of long chain AHLshinders bacterial quorum sensing activity that would otherwise serve toexacerbate γ-proteobacteria infection in a subject.

The screening assays of the invention allow the identification of agentsthat modulate γ-proteobacterium AHL acylase activity by increasing AHLacylase gene expression and/or AHL acylase activity, thereby increasingthe degradation of long chain AHLs. Where an agent increases (ordecreases) AHL acylase gene expression, the increased (or decreased)expression results in increased (or decreased) AHL acylase levels andAHL acylase activity. As such, an agent that increases AHL acylase geneexpression and/or AHL acylase activity, when administered to a subject,can decrease virulence of the bacteria expressing the AHL acylase in thesubject. The subject can be any subject affected by γ-proteobacteriainfections, for example, an immunocompromised human subject, or anindividual suffering from cystic fibrosis or other disease that rendersthe subject susceptible to infection by γ-proteobacteria.

According to a method of the invention, a change in AHL acylase activitycan be detected using any assay suitable for measuring AHL acylaseactivity, including, for example, assays that can measure AHL levelsand/or AHL breakdown products such as a fatty acid and/or HSL in thesample (or an aliquot or fraction of the sample). Examples of methodsuseful for measuring a change in AHL acylase activity are provided,including mass spectroscopy and thin layer chromatography, which candetect levels of the AHL acylase substrate (AHL) and/or product (e.g.,HSL), and additional methods for measuring AHL acylase activity (e.g.,enzyme kinetics assays) are well known in the art. In addition, abiological assay based, for example, on quorum sensing activity of amedium containing the AHL acylase before and after contact with a testagent can be used to measure a change in AHL acylase activity.

The screening assays of the invention also allow the identification ofan agent that decreases AHL acylase gene expression and/or AHL acylaseactivity, thereby decreasing breakdown of long chain AHLs. For example,certain bacteria utilize quorum sensing to regulate the production ofplant protecting antifungal compounds (e.g., in suppressing fungaldisease that impacts wheat roots). Agents that reduce or inhibit AHLdegradation can be useful for boosting and improving quorum sensing ofsuch beneficial bacteria, which often are used in “biocontrol”agricultural regimes.

Acyl-homoserine lactones are chemically inactivated via alkalinehydrolysis, yielding the cognate acyl-homoserine (41), but are stablefor weeks or months at pH values of 5 to 6 (34). AHLs are also subjectto biological inactivation (see FIG. 1). Similar to abiotic ringhydrolysis, acyl-homoserine can be generated by acyl-HSL lactonasesencoded by Bacillus cereus (and its close relatives) and byAgrobacterium tumefaciens (6, 8, 22, 32, 46). None of these strains hasbeen found to further degrade the molecule, and no net oxidation occursduring this inactivation reaction. More complete degradation can occuras evidenced by an Arthrobacter soil isolate that utilizes theacyl-homoserine degradation products of AHL ring hydrolysis reactions(10). In another mechanism of AHL inactivation, the amide bond of AHL iscleaved by AHL acylases during the utilization of quorum signals asgrowth nutrients by Variovorax and Ralstonia species (21, 23).Homoserine lactone is released as a product of these reactions and theacyl-moiety is further metabolized as an energy substrate (19, 21). Whena gene encoding an AHL acylase from Ralstonia (AiiD) was expressed in E.coli and in P. aeruginosa, it effectively inactivated endogenouslyproduced AHL quorum signals and quenched quorum sensing in P. aeruginosa(23).

A close homologue of the Ralstonia AHL acylase was identified in P.aeruginosa PAO1 and in the genomes of several other sequencedpseudomonads (23), which often produce AHLs and engage in quorumsensing. As disclosed herein, the P. aeruginosa PAO1, as well as a newlyisolated Pseudomonas strain, PAI-A, utilized 3OC12HSL as an energysource, and exhibited growth on long chain acyl AHLs. Further, P.aeruginosa PvdQ, which is a Ralstonia AiiD homolog, was found to haveAHL acylase activity with respect to long chain, but not short chain,AHLs.

The soil pseudomonad, strain PAI-A, was enriched and isolated, based onits ability to degrade 3OC12HSL, which is a virulence factor that isproduced by and used as a dedicated signal in the quorum sensingphysiology of the opportunistic pathogen P. aeruginosa. Subsequently, itwas ascertained that two clinical strains of P. aeruginosa were capableof degrading and growing on 3OC12HSL and other long-acyl AHLs (seeExample 1). None of the pseudomonads examined degraded either C4HSL, theother distinct AHL quorum signal produced by P. aeruginosa, or othershort acyl AHLs tested. Although closely related to P. aeruginosa,strain PAI-A is a separate species, as it did not produce pigments,acyl-HSLs, or grow at 42° C. or anaerobically with nitrate as terminalelectron acceptor.

Using a refined LC/APCI-MS technique, the soil and clinical pseudomonadswere shown to degrade AHLs via an HSL-releasing activity, indicatingthat these pseudomonads use an AHL acylase in the initial step of AHL,degradation, similar to the mechanism described in Variovorax andRalstonia isolates (21, 23). P. aeruginosa accumulated HSL as atransient intermediate during degradation of long chain AHLs, and theHSL was subsequently delactonized to form homoserine, which was consumed(see FIG. 4). Since the culture pH was well controlled in order topreclude the chemical hydrolysis of lactone ring, the observed HSLdegradation was due to a biological, and not to an abiotic, hydrolysisevent. Enzymes with HSL lactonase activity are expressed in fungal andmammalian biota (15, 17). However, neither HSL nor homoserine was usedas an energy or nitrogen source by P. aeruginosa, as they are byVariovorax and Arthrobacter species (10, 21). The HSL lactonase andhomoserine degrading activities may serve as detoxification mechanisms,since both of these compounds are known to be toxic to diverse biota(10, 14, 15, 45).

There are notable differences between AHL utilization by thepseudomonads and Variovorax paradoxus and Ralstonia strain XJ12B.Ralstonia was reported to degrade and grow equally rapidly with shortand long acyl AHLs (23). Variovoras paradoxus was reported to utilizethe entire spectrum of short and long acyl AHLs tested, and grew mostrapidly with N-3-oxohexanoyl-L-homoserine lactone (3OC6HSL, see ref.21). Moreover, Variovorax exhibited molar growth yields thatcorresponded well with the acyl length of a given AHL. As disclosedherein, however, pseudomonads did not degrade AHLs with acyl side chainsshorter than eight carbons, and no correspondence was observed betweenmolar growth yields and AHL acyl side chain lengths (Table 1; seebelow), although a correspondence was observed when the cells were grownwith long chain fatty acids.

Acyl homoserine lactone utilization by P. aeruginosa exhibited anotherkey difference from Variovorax, Ralstonia, and even Pseudomonas strainPAI-A. When cultures not previously grown on AHL were inoculated intolong acyl AHL containing media, it generally took one to three weeksbefore logarithmic growth commenced. However, no lag was observed whenAHL grown cells were subcultured into such medium. This adaptation doesnot appear to reflect a stable mutation, as long lags were againobserved if the subculturing process was punctuated with a transfer intoor onto media containing a different energy substrate. The long initiallag time suggests that AHL degradation by P. aeruginosa is notimmediately induced by the quorum signal, and is not controlled as afunction of the catabolic needs of the cell or by cell starvation.

Agrobacterium tumefaciens degrades N-3-oxo-octanoyl-L-homoserine lactone(3OC8HSL), its AHL quorum signal, during early stationary phase (46).The disclosure that P. aeruginosa can degrade one, but not the other, ofits two AHL quorum signals has revealed a previously undescribed AHLdegradation apparatus. Signal decay, in addition to providing utilizablenutrients, can be involved in the regulation of theLasR/LasI/3OC12HSL-controlled quorum sensing regulon. The two principalAHL quorum signals of P. aeruginosa, C4HSL and 3OC12HSL, are present inthe sputum of cystic fibrosis patients and in laboratory biofilms atratios quite different from those encountered in planktonic, liquidgrown cultures. Sputum and biofilm samples contained significantlyhigher levels of C4HSL with respect to 3OC12HSL. Based on the presentresults, the different levels of the short chain C4HSL and long chain3OC12HSL are likely due to biochemical turnover of the long chain AHL.

The Ralstonia AiiD enzyme inactivates long chain and short chain AHLs(23). Heterologous expression of PvdQ, which is the closest homolog ofthe Ralstonia acylase encoded by P. aeruginosa, in E. coli conferredAHL-acylase activity specific towards long acyl, but not short acyl,chain, AHLs (FIG. 5). Expression of the PvdQ gene in P. aeruginosa iswell regulated. The PvdQ gene was identified as being a late responderto the 3OC12HSL quorum sensing circuit (see ref 43, which refers to thePvdQ gene as QSC 112a and QSC 112b), although gene microarray studieshave not provided further support for this observation (37, 42). P.aeruginosa gene PvdQ is iron-regulated (FUR-repressed), and appears tobe involved in pyoverdine biosynthesis, based on evidence from bothmicroarray and mutagenesis studies (18, 29). The effects of theconstitutive expression of plasmid-encoded PvdQ in strain PAO1 wereexamined to gather information on its complicated control mechanism.Remarkably, 3OC12HSL did not accumulate during growth of P. aeruginosaconstitutively expressing PvdQ in a rich medium, in striking contrast tothe behavior of the wild type, which produced micromolar amounts of thisquorum signal (FIG. 6).

Two PvdQ knockout mutants grew with 3OC12HSL as a sole energy source,suggesting that another enzyme confers the AHL growth phenotype instrain PAO1. Although some contribution of PvdQ to AHL-utilizationcannot be ruled out, it is more likely, as previously suggested, thatthis protein is involved in an editing reaction during the maturation ofthe pyoverdine siderophore (18, 29). However, 3OC12HSL also may besubject to inadvertent biochemical degradation by PvdQ during times ofpyoverdine expression. The gene encoding the acylase AHL-utilizationphenotype of P. aeruginosa has not yet been described.

P. aeruginosa mutants, in which the PvdQ gene was knocked out, were ableto grow in long chain AHL-acylases, as compared to E. coli expressingPvdQ, which catalyzed the inactivation of long chain AHLs and therelease of HSL. In addition, P. aeruginosa engineered to express PvdQdid not accumulate long chain (3OC12HSL) quorum signals. These resultsconfirm that the degradation of AHL acylase was due to increased AHLacylase activity, and indicate that methods for increasing AHL acylaseactivity can be useful for decreasing virulence by degrading quorumsensing AHLs.

Accordingly, the present invention provides methods of identifying anagent that modulates γ-proteobacterium long chain AHL acylase activityby contacting at least one sample containing the AHL acylase and a longchain AHL with a test agent, under conditions suitable for AHL acylaseactivity, and detecting a change in AHL acylase activity in the presenceof the test agent as compared to the AHL acylase activity in the absenceof the test agent, wherein a change in AHL acylase activity identifiesthe test agent as an agent that modulates the γ-proteobacterium longchain AHL acylase activity. As used herein, the term “modulate” means toincrease or decrease. As used herein, the term “long chain AHL” means anAHL having a fatty acid moiety containing eight or more carbon residues(e.g., C8HSL, C10HSL, 3OC12HSL, C14HSL). In comparison, the term “shortchain AHL” means an AHL having a fatty acid moiety containing seven orfewer carbon residues (e.g. C4HSL, C6HSL, 3OC6HSL, C7HSL).

An AHL acylase useful in a method of the invention can be any AHLacylase that degrades long chain, but not short chain, AHLs. Generally,the AHL acylase is a γ-proteobacterium AHL acylase, for example, aPseudomonas PvdQ AHL acylase, which has an amino acid sequence as setforth in SEQ ID NO:2 (encoded by SEQ ID NO:1), or a Pseudomonas PA1032AHL acylase, which has an amino acid sequence as set forth in SEQ IDNO:4 (encoded by SEQ ID NO:3) or SEQ ID NO:5 (encoded by nucleotides 19to 2544 of SEQ ID NO:3; alternative initiator methionine residue atnucleotides 19 to 21 of SEQ ID NO:3). The term “AHL acylase activity” isused herein to refer to the enzymatic activity of an AHL acylase,including the rate of long chain AHL degradation (breakdown) by an AHLacylase. AHL acylase activity can be measured using methods as disclosedherein or methods of determining enzyme kinetics as otherwise known inthe art, such that an increase or decrease in AHL activity due tocontact with a test agent can be identified. Reference herein to “AHLacylase gene expression” means transcription and translation, ortranslation, of an AHL acylase coding sequence such that the AHL acylaseprotein is produced. Increased AHL acylase gene expression results inincreased AHL acylase levels produced by a γ-proteobacterium, anddecreased AHL acylase gene expression results in decreased AHL acylaselevels produced by a γ-proteobacterium. An agent that increases AHLacylase gene expression, for example, can act by inducing transcriptionof a γ-proteobacterium AHL acylase gene or by derepressing theγ-proteobacterium AHL acylase gene, or increasing translation of an AHLacylase coding sequence (e.g., mRNA). In this respect, it should berecognized that iron starvation has been reported to up-regulate thelocus comprising AHL acylase gene, PvdQ, in Pseudomonas, and that a“late” response is observed in Pseudomonas following contact with3OC12HSL, suggesting 3OC12HSL directly or indirectly induces AHL acylasegene expression; these aspects of AHL acylase gene regulation are notconsidered to be encompassed within the present methods.

The methods of the invention provide screening assays useful fordetermining whether a test agent can modulate the activity of aγ-proteobacterium AHL acylase. As used herein, the term “test agent”means any compound that is being examined for the ability to modulateAHL acylase activity. A test agent (and an agent that modulates AHLacylase activity identified by a method of the invention) can be anytype of molecule, including, for example a peptide, a polynucleotide, anantibody, a glycoprotein, a carbohydrate, a small organic molecule, or apeptidomimetic.

The term “polynucleotide” is used broadly herein to mean a sequence oftwo or more deoxyribonucleotides or ribonucleotides that are linkedtogether by a phosphodiester bond. As such, the term “polynucleotide”includes RNA and DNA, which can be an isolated naturally occurringpolynucleotide or portion thereof or a synthetic polynucleotide, and canbe single stranded or double stranded, as well as a DNA/RNA hybrid. Apolynucleotide agent (or test agent) can contain nucleoside ornucleotide analogs, or a backbone bond other than a phosphodiester bond.In general, the nucleotides comprising a polynucleotide are naturallyoccurring deoxyribonucleotides, such as adenine, cytosine, guanine orthymine linked to 2′-deoxyribose, or ribonucleotides such as adenine,cytosine, guanine or uracil linked to ribose. However, a polynucleotidealso can contain nucleotide analogs, including non-naturally occurringsynthetic nucleotides or modified naturally occurring nucleotides. Suchnucleotide analogs are well known in the art and commercially available,as are polynucleotides containing such nucleotide analogs (Lin et al.,Nucl. Acids Res. 22:5220-5234, 1994; Jellinek et al., Biochemistry34:11363-11372, 1995; Pagratis et al., Nature Biotechnol. 15:68-73,1997, each of which is incorporated herein by reference).

The covalent bond linking the nucleotides of a polynucleotide generallyis a phosphodiester bond. However, the covalent bond also can be any ofnumerous other bonds, including a thiodiester bond, a phosphorothioatebond, a peptide-like bond or any other bond known to those in the art asuseful for linking nucleotides to produce synthetic polynucleotides(see, for example, Tam et al., Nucl. Acids Res. 22:977-986, 1994; Eckerand Crooke, BioTechnology 13:351360, 1995, each of which is incorporatedherein by reference). The incorporation of non-naturally occurringnucleotide analogs or bonds linking the nucleotides or analogs can beparticularly useful where the polynucleotide is to be exposed to anenvironment that can contain a nucleolytic activity, including, forexample, a tissue culture medium or upon administration to a livingsubject, since the modified polynucleotides can be less susceptible todegradation.

A polynucleotide comprising naturally occurring nucleotides andphosphodiester bonds can be chemically synthesized or can be producedusing recombinant DNA methods, using an appropriate polynucleotide as atemplate. In comparison, a polynucleotide comprising nucleotide analogsor covalent bonds other than phosphodiester bonds generally will bechemically synthesized, although an enzyme such as T7 polymerase canincorporate certain types of nucleotide analogs into a polynucleotideand, therefore, can be used to produce such a polynucleotiderecombinantly from an appropriate template (Jellinek et al., supra,1995).

Peptides also can be useful as test agents. The term “peptide” is usedbroadly herein to refer to a molecule containing two or more amino acidsor amino acid analogs (or modified forms thereof) linked by peptidebonds. As such, peptide test agents (or agents) can contain one or moreD-amino acids and/or L-amino acids; and/or one or more amino acidanalogs, for example, an amino acid that has been derivatized orotherwise modified at its reactive side chain. In addition, one or morepeptide bonds in the peptide can be modified, and a reactive group atthe amino terminus or the carboxy terminus or both can be modified.Peptides containing D-amino acids, or L-amino acid analogs, or the like,can have improved stability to a protease, an oxidizing agent or otherreactive material the peptide may encounter in a biological environment.Further, the stability of a peptide agent (or test agent) can beimproved by generating (or linking) a fusion protein comprising thepeptide and a second polypeptide (e.g., an Fc domain of an antibody)that increases the half-life of the peptide agent in vivo. Peptides alsocan be modified to have decreased stability in a biological environment,if desired, such that the period of time the peptide is active in theenvironment is reduced.

Antibodies provide an example of peptides useful as test agents in ascreening assay of the invention. As used herein, the term “antibody” isused in its broadest sense to include polyclonal and monoclonalantibodies, as well as antigen binding fragments of such antibodies.Antibodies are characterized, in part, in that they specifically bind toan antigen, particularly to one or more epitopes of an antigen. The term“binds specifically” or “specific binding activity” or the like, whenused in reference to an antibody, means that an interaction of theantibody and a particular epitope has a dissociation constant of atleast about 1×10⁻⁶ M, generally at least about 1×10⁻⁷ M, usually atleast about 1×10⁻⁸ M, and particularly at least about 1×10⁻⁹ M or1×10⁻¹⁰ M or less. As such, Fab, F(ab′)₂, Fd and Fv fragments of anantibody that retain specific binding activity are included within thedefinition of an antibody.

The term “antibody” as used herein includes naturally occurringantibodies as well as non-naturally occurring antibodies, including, forexample, single chain antibodies, chimeric, bifunctional and humanizedantibodies, as well as antigen-binding fragments thereof. Suchnon-naturally occurring antibodies can be constructed using solid phasepeptide synthesis, can be produced recombinantly or can be obtained, forexample, by screening combinatorial libraries consisting of variableheavy chains and variable light chains (see Huse et al., Science246:1275-1281, 1989, which is incorporated herein by reference). Theseand other methods of making, for example, chimeric, humanized,CDR-grafted, single chain, and bifunctional antibodies are well known(Winter and Harris, Immunol. Today 14:243-246, 1993; Ward et al., Nature341:544-546, 1989; Harlow and Lane, Antibodies. A laboratory manual(Cold Spring Harbor Laboratory Press, 1999); Hilyard et al., ProteinEngineering. A practical approach (IRL Press 1992); Borrabeck, AntibodyEngineering, 2d ed. (Oxford University Press 1995); each of which isincorporated herein by reference)

A screening assay of the invention is practiced by contacting a samplethat contains (or to which can be added) an AHL acylase and/or a longchain AHL, under conditions suitable for AHL acylase activity. Suchconditions are exemplified herein (see Examples 1 and 2), and include,for example, an appropriate concentration of iron ions sufficient forAHL acylase activity, as well as appropriate buffer conditions(including pH), salt concentration (e.g., physiological), and otherconditions, which can be selected based on whether the assay isperformed in a cell free format or is performed in a cell based assay.

As disclosed herein, a screening assay of the invention can be performedin vitro (e.g., in a cell free system using purified or partiallypurified components) or in a cell (e.g., in a cell or tissue culturesystem). Where the method is performed in vitro, the AHL acylase can bea purified naturally occurring Ail, acylase, which can be obtained, forexample, from an extract comprising a Pseudomonas species (e.g., P.aeruginosa), or can be a synthetic AHL acylase prepared, for example,using an in vitro translation or coupled transcription/translationreaction using a polynucleotide as set forth in SEQ ID NO:1 or SEQ IDNO:3, including nucleotides 1 to 2544 or nucleotides 19 to 2544 of SEQID NO:3, as a template. Where the method is performed as a cell basedassay, the sample can be a cell sample, wherein the AHL acylase isexpressed in the cell. The cell can be a γ-proteobacterium, in which theAHL acylase is expressed in nature (e.g., a Pseudomonas species such asPseudomonas aeruginosa), can be a host cell or tissue sample that isinfected with γ-proteobacteria (e.g., a biopsy sample from a subjectinfected with the bacteria), or can be a cell that has been geneticallymodified to express a polynucleotide encoding a γ-proteobacterium AHLacylase (e.g., a host cell transformed, transfected or transduced with apolynucleotide encoding the AHL; see Example 2).

Where a test agent is identified as having γ-proteobacterium AHL acylasemodulating activity, a screening assay of the invention can furtherinclude a step of determining an amount by which the agent increases ordecreases γ-proteobacterium AHL acylase modulating activity. Forexample, where an agent is identified that increases AHL acylaseactivity in a cell, a method of the invention can further includedetermining an amount by which the agent increases AHL acylase activityabove a basal level. Such an agent can be identified by measuring theamount of AHL acylase activity in a single sample both before adding thetest agent and after adding the test agent, or can be identified forexample, using two samples, wherein one sample serves as a control (notest agent added) and the other sample includes the test agent. As such,a method of the invention provides a means to obtain agents or panels ofagents that variously modulate AHL acylase activity.

A screening assay of the invention also provides a means to determine anamount of a particular agent useful for effecting a desired level of AHLacylase activity. Such a method can be performed by contacting aliquotsof a sample with different amounts of the same or different test agentsor different amounts of the same or different agents previouslyidentified as having AHL acylase modulating activity. As such, themethods of the invention can be used to confirm that an agent believedto have a particular activity, in fact, has the activity, thus providinga means, for example, to standardize the activity of the agent.

The screening method of the invention is readily adaptable to highthroughput format, thus allowing for the screening, in parallel, of oneor more test agents using one or more samples, wherein the agents and/orsamples independently are the same or different. As such, the methodallows for testing one or more concentrations of one or more test agentsto identify a concentration of an agent particularly useful formodulating a γ-proteobacterium AHL acylase activity. Further, the methodallows for examining several same test agents on one or a plurality ofsame samples, thus providing a means to obtain statistically significantresults. In various aspects, the high throughput format can be used forscreening one or a plurality of cell sample(s) taken from a subjecthaving a γ-proteobacterium (e.g., P. aeruginosa) infection with one or aplurality of the same (e.g., different concentrations) or different testagents, to identify an agent and/or concentration of agent that is bestsuited, for example, for increasing AHL acylase activity in the patient,which increases the rate of breakdown of long chain AHLs produced by theγ-proteobacterium, thus providing an agent that reduces or inhibitsquorum sensing activity by the infecting bacteria, thereby amelioratingthe infection.

When performed in a high throughput (or ultra-high throughput) format,the method can be performed on a solid support (e.g., a microtiterplate, a silicon wafer, or a glass slide), wherein samples to becontacted with a test agent are positioned such that each is delineatedfrom each other (e.g., in wells). Any number of samples (e.g., 96, 1024,10,000, 100,000, or more) can be examined in parallel using such amethod, depending on the particular support used. Where samples arepositioned in an array (i.e., a defined pattern), each sample in thearray can be defined by its position (e.g., using an x-y axis), thusproviding an “address” for each sample. An advantage of using anaddressable array format is that the method can be automated, in wholeor in part, such that reagents (e.g., test agents) can be dispensed in(or removed from) specified positions at desired times, and samples (oraliquots) can be monitored for AHL acylase activity.

When used in a high throughput format, a method of the inventionprovides a means to conveniently screen combinatorial libraries of testagents, which can be a library of random test agents, biased test agents(see, for example, U.S. Pat. No. 5,264,563, which is incorporated hereinby reference), or variegated test agents (see, for example, U.S. Pat.No. 5,571,698, which is incorporated herein by reference), in order toidentify those agents that can modulate γ-proteobacterium AHL acylaseactivity. Methods for preparing a combinatorial library of moleculesthat can be screened for AHL acylase modulating activity are well knownin the art and include, for example, methods of making a phage displaylibrary of peptides, which can be constrained peptides (see, forexample, U.S. Pat. No. 5,622,699; U.S. Pat. No. 5,206,347; Scott andSmith, Science 249:386-390, 1992; Markland et al., Gene 109:13 19, 1991;each of which is incorporated herein by reference); a peptide library(U.S. Pat. No. 5,264,563, which is incorporated herein by reference); alibrary of peptide derivative compounds such as a hydroxamate compoundlibrary, reverse hydroxamate compound library, a carboxylate compoundlibrary, thiol compound library, a phosphinic peptide library, orphosphonate compound library (see, for example, Dive et al., Biochem.Soc. Trans. 28:455-460, 2000; Ye and Marshall, “Peptides: The Wave ofthe Future” (Lebl and Houghten, ed.; American Peptide Society, 2001),each of which is incorporated herein by reference); a peptidomimeticlibrary (Blondelle et al., Trends Anal. Chem. 14:83 92, 1995, which isincorporated herein by reference); a nucleic acid library (O'Connell etal., Proc. Natl. Acad. Sci., USA 93:5883-5887, 1996; Tuerk and Gold,Science 249:505-510, 1990; Gold et al., Ann. Rev. Biochem. 64:763-797,1995; each of which is incorporated herein by reference); anoligosaccharide library (York et al., Carb. Res. 285:99-128, 1996; Lianget al., Science 274:1520 1522, 1996; Ding et al., Adv. Expt. Med. Biol.376:261269, 1995; each of which is incorporated herein by reference); alipoprotein library (de Kruif et al., FEBS Lett. 399:232 236, 1996,which is incorporated herein by reference); a glycoprotein or glycolipidlibrary (Karaoglu et al., J. Cell Biol. 130:567 577, 1995, which isincorporated herein by reference); or a chemical library containing, forexample, drugs or other pharmaceutical agents (Gordon et al., J. Med.Chem. 37:1385-1401, 1994; Ecker and Crooke, BioTechnology 13:351-360,1995; each of which is incorporated herein by reference).

As disclosed herein, a method of the invention can be performed using asample comprising a cell, tissue or biologic fluid obtained from asubject having a γ-proteobacterium infection (e.g., a subject having aPseudomonas infection). The subject can be any subject susceptible toinfection by the γ-proteobacterium, including any vertebrate such as amammal (e.g., a human subject infected by P. aeruginosa or by V.cholerae). As such, the methods provide a means to identify an agentthat is useful for ameliorating a pathology due to a γ-proteobacteriainfection in a subject (e.g., a Pseudomonas, Vibrio, Legionellales,Azotobacter, or Enterobacteriales infection). As used herein, the term“ameliorate” means that signs and/or symptoms of a γ-proteobacteriainfection in a subject are reduced (lessened). Such a method can beperformed, for example, by administering to the subject an agent thatmodulates AHL acylase activity of a γ-proteobacterium by degradation oflong chain AHLs, thereby preventing autoinduction which would otherwiseoccur due to quorum bacteria reaching threshold concentrations. As such,immunocompromised subjects, subjects afflicted with cystic fibrosis,burn patients, and any other subject particularly susceptible toinfection by an opportunistic γ-proteobacterium can benefit fromtreatment with an agent identified according to a method of theinvention.

Amelioration of a γ-proteobacteria infection can be identified using anyassay generally used to monitor the clinical signs or the symptoms ofthe particular disorder. For example, P. aeruginosa generally infectsthe lungs and, therefore, the skilled clinician would know that asubject having such an infection can be monitored by testing a sputumsample of the subject for P. aeruginosa, wherein decreased amounts oractivity of the P. aeruginosa are indicative of amelioration. Similarly,the cardinal signs of infection (e.g., fever) generally are observed insuch infected subjects and can be monitored using routine and well knownmethods. In addition, amelioration can be identified by the subjectindicating that he or she feels better following treatment with an agentidentified according to a method of the invention.

Where the agent is to be used for a therapeutic method, it can beformulated in a form suitable for administration to a subject, forexample, as a pill or a liquid, and can be administered, for example,orally, by injection, or via inhalation. Accordingly, compositions,including medicaments, useful for treating a subject infected with aγ-proteobacterium (e.g., P. aeruginosa) are provided. A composition foradministration to a living subject generally includes formulating theagent in a pharmaceutically acceptable composition. Such compositionsare well known in the art and include, for example, aqueous solutionssuch as water or physiologically buffered saline or other solvents orvehicles such as glycols, glycerol, oils such as olive oil or injectableorganic esters. The composition also can contain physiologicallyacceptable compounds that act, for example, to stabilize or to increasethe absorption of the agent. Such physiologically acceptable compoundsinclude, for example, carbohydrates, such as glucose, sucrose ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins or other stabilizers orexcipients.

One skilled in the art would know that the choice of a composition,including a physiologically acceptable compound, depends, for example,on the physico-chemical characteristics of the agent to be administered,and on the route of administration of the composition, which can be, forexample, orally or parenterally such as intravenously, and by injection,intubation, inhalation, or other such method known in the art. Thecomposition also can contain one or more additional reagents, including,for example, nutrients or vitamins or, where the composition isadministered for a therapeutic purpose, a diagnostic reagent ortherapeutic agent relevant to the disorder being treated.

The composition can be administered to a subject by any of variousroutes including, for example, orally or parenterally, such asintravenously, intramuscularly, subcutaneously, intraorbitally,intracapsularly, intraperitoneally, intrarectally, intracisternally orby passive or facilitated absorption through the skin using, forexample, a skin patch or transdermal iontophoresis, respectively.Furthermore, the composition can be administered by injection,intubation, orally or topically, the latter of which can be passive, forexample, by direct application of an ointment, or active, for example,using a nasal spray or inhalant, in which case one component of thecomposition is an appropriate propellant. Inhalation can be aparticularly useful means of administration where the γ-proteobacteriuminfection is in the lungs (e.g., a P. aeruginosa infection).

The following examples are intended to illustrate but not limit theinvention.

Example 1 Isolation and Growth of Pseudomonas Strains

This example illustrates how different Pseudomonas bacterial strainsresponded when isolated and given only 3OC12HSL-containing medium as anenergy source. The bacterial strains used were: Pseudomonas strain PAI-A(isolation described below); P. aeruginosa PA 14 (obtained from DianneNewman of Caltech); P. aeruginosa PAO1 and QSC 112a (obtained from E.Peter Greenberg of the University of Iowa; 43); P. aeruginosa PAO1 andan in-frame PvdQ-deletion/Gen^(R)-cassette-replacement mutant of thisPAO1 Denver strain (both obtained from Michael Vasil of the Universityof Colorado Health Sciences Center; 29); P. aeruginosa PAO1 containingpPvdQ-Nde (see below), a constitutive PvdQ expression vector derivedfrom pUCP-Nde (4); E. coli DH5α carrying pUCP18-Nde (obtained fromCiaran Cronin of the University of California, San Francisco); E. coliBL21PRO containing pPvdQ-PROTet, a pPROTet.E133-derived tet-induciblePvdQ expression vector encoding tetracycline and spectinomycinresistance (see below); and E. coli BL21PRO containing the autonomouslyreplicating plasmid, pPROTet.E133 (Clontech).

Media and Growth Conditions.

LB, amended with antibiotics when appropriate, was used for growth andmaintenance of all strains unless otherwise stated. For the3OC12HSL-dependent enrichment of strain PAI-A and other growthexperiments performed on this strain, modified “MES 5.5” defined-mediumwas used to enrich and study Variovorax paradoxus VAI-C (21). Thesemodifications to the medium included buffering with 5 mM3-(N-morpholino)-propanesulfonic acid (MOPS) at pH 7.2 and the additionof sodium sulfate (14 g liter⁻¹) and magnesium chloride (4 g liter⁻¹).For growth experiments with P. aeruginosa, “MES 5.5” defined medium wasused as described previously (21) with the exception that it containedsodium sulfate, not sulfite, as S-source, a typographical error in thereported recipe. Unless otherwise noted, the medium was buffered to a pHof 5.5 with 5 mM 2-(N-morpholino)-ethanesulfonic acid (MES).Ammonium-free “MES 5.5” basal medium was used to examine the utilizationof AHLs and HSL as potential nitrogen sources. 100 mM stock solutions ofAHLs were prepared by dissolving AHLs in ethyl acetate that had beenacidified with glacial acetic acid (0.01% v/v), and stocks were storedat −20° C. AHLs used in these studies were:N-3-oxododecanoyl-L-homoserine lactone (3OC12HSL; Quorum Sciences Inc.,Iowa City Iowa), N-3-oxohexanoyl-L-homoserine lactone (3OC6HSL; Sigma),and from Fluka: N-3-butanoyl-DL-homoserine lactone (C4HSL),N-3-hexanoyl-DL-homoserine lactone (C6HSL), N-3-heptanoyl-DL-homoserinelactone (C7HSL), N-3-octanoyl-DL-homoserine lactone (C8HSL),N-3-dodecanoyl-DL-homoserine lactone (C10HSL),N-3-dodecanoyl-DL-homoserine lactone (C12HSL),N-3-tetradecanoyl-DL-homoserine lactone (C14HSL). For growthexperiments, the AHL was dispensed into sterile tubes, the ethyl acetatewas removed by evaporation under a stream of sterile air, and sterilemedium was added to the dried AHL that remained. Stocks of L-HSL (Sigma)were prepared just prior to their use from well-desiccated reagentstored at −20° C. That homoserine contamination was not present wasverified via thin layer chromatography and ninhydrin staining (16).Cells were grown in 5 ml of medium in 18-mm tubes with shaking at 37° C.unless otherwise noted. AHL molecules are stable for approximately 30days under the conditions of low pH in our defined medium (9, 34).Unless noted, all other reagents were of reagent grade.

Enrichment and Isolation Procedures.

Turf soil was collected in May of 2000 at the University of Iowa. Thesoil was disrupted and dispersed with a metal spatula, and remaininglarge particles were removed. One hundred milligrams of the soil wereadded to 5 ml of the vitamins replete, ammonium-replete enrichmentmedium containing 1 mM 3OC12HSL as a sole energy source (see above).After 2 days of incubation with shaking at 37° C., a 1% (vol/vol)transfer was made into like medium. This culture was incubated withoutagitation at room temperature for 3 months after which the culture wastransferred once more into 3OC12HSL-containing medium before beingstreaked for isolation on rich media. Because 3OC12HSL is not soluble atthe concentrations employed for growth, isolation was on LB agar withsubsequent verification of the AHL-degradation phenotype in the definedliquid medium.

Growth Studies.

Optical density measurements were performed at 600 nm using a Spectronic20 spectrophotometer. AHLs with side chains of greater than 6 carbons inlength were poorly soluble, so the ethyl acetate carrier was evaporatedin the glass tube such that a uniform coating of AHL was beneath thespectrophotometer's light path. When care was taken to vortex tubesgently, the changes in optical density reflecting growth could bemonitored accurately. Molar growth yields were determined in the definedmedia containing the indicated substrate at a final concentration of 1or 2 mM. For both Pseudomonas PAI-A and P. aeruginosa PAO1, factors forconverting optical density to cell dry mass were determined by growingcells in media containing succinate as the energy source and NH₄Cl asthe nitrogen source, washing the cells with 50 mM ammonium acetatebuffer (pH 5.5), and drying cell samples to a constant weight. Suchdeterminations were made in quadruplicate.

Enrichment and Isolation of a Bacterium that Utilizes3-oxododecanoyl-HSL as a Sole Energy Source.

An enrichment culture using a 3OC12HSL-containing minerals and vitaminsmedium became turbid within 48 hours after inoculation with turf soil.No growth was evident in a control lacking energy nutrient. The cellswere rods of uniform morphology and were well dispersed in the medium.They did not form clumps, a pellicle, or attach to the glass at theair-medium interface. When the culture was streaked on LB agar mediumfor isolation, a single, uniform colony morphotype was observed. Purecultures were obtained after several successive streaks from singlecolony picks. Growth of a representative isolate, designated strainPAI-A, was confirmed in the 3OC12HSL-containing liquid medium.

Examination of Pseudomonas aeruginosa Strains for the Ability to Utilize3OC12HSL.

Two clinical strains of P. aeruginosa, PAO1 and PA14, were examined forthe ability to utilize 3OC12HSL in defined, ammonia-replete media atboth pH 5.5 and 7.2. Both strains grew rapidly at both pHs usingsuccinate as a sole energy source. Although initially it appeared as ifneither would utilize the quorum signal as an energy nutrient, thestrains began to grow exponentially with a doubling time ranging from11-25 days after several weeks incubation. The length of the initial lagphase in cultures inoculated using naive cells (those not previouslygrown on AHL) was highly variable, ranging from 10 to 30 days.Curiously, AHL-grown cells that were transferred directly intoAHL-containing media did not show significant lags in growth, but thosetransferred and grown in media containing a different energy substratefollowed by re-introduction into AHL both re-exhibited long lag phases.The issues underlying the long lags exhibited by naive cells and theirsubsequent adaptation to growth on AHLs have not been further clarified.

Pseudomonas PAI-A and P. aeruginosa PAO1 Degrade and Utilize Long AcylAHLs.

Strains PAI-A and PAO1 grew on a number of AHLs, but no growth wasobserved with AHLs with acyl side chains shorter than 8 carbons (Table1). When provided with 1 mM C4HSL as a co-substrate in3OC12HSL-containing media, PAI-A and PAO-1 did not degrade detectableamounts of the short chain AHL or exhibit any C4HSL-dependentstimulation of their growth yields. Optical density to “dry weightbiomass” conversion factors were determined to be, at an OD₆₀₀ of 1.0:346±7 μg·ml⁻¹ for strain PAO-1, and 337±8 μg·ml⁻¹ for strain PAI-A. Thedoubling times of strains PAI-A and PAO1 were comparable for manysubstrates (Table 1). P. aeruginosa PAO1 utilized both the D-forms andL-forms of AHLs, as determined by substrate disappearance and comparisonof the molar yields on L-forms and DL-forms. Curiously, no increase inmolar growth yield was observed as a function of AHL acyl lengths, i.e.when comparing growth on C10HSL, C12HSL, and C14HSL (for contrast, seeFIG. 5 of (21)). The AHL molar growth yields for strain (str.) PAO-1were only 49% to 67% of that achieved during its growth on thecorresponding fatty acids (Table 1). Growth on fatty acids revealed theexpected, incremental increase in molar yield as a function of increasedacyl length. Neither strain PAI-A nor PAO1 used HSL as sole orsupplementary energy source.

Strains PAI-A and PAO1 Release HSL as an Initial Product of AHLDegradation.

Thin layer chromatography of clarified reaction fluids, harvested fromdense cell suspensions of strains PAI-A and PAO1 incubated with 25 mMC12HSL, revealed the AHL-dependent release of ninhydrin-reactivematerials. These had the same yellow and purple staining characteristicsand migration characteristics as authentic HSL and homoserine,respectively (data not presented). In contrast, cell-free, AHL-free, andcell and AHL-free controls did not produce ninhydrin reactive materialsafter similar incubation periods. The TLC data suggests that bothstrains catalyze the initial step of AHL degradation via anHSL-releasing acylase. Because analyses of biological AHL degradationsare less ambiguous at pH 5.5 than they are at pH 7.2, and because strainPAI-A does not grow on AHLs at pH 5.5, P. aeruginosa was chosen forfurther experiments.

APCI LC/MS analyses confirmed that P. aeruginosa PAO1 releases HSL andhomoserine as AHL degradation products. A representative chromatogram ofcell-free fluid from a C10HSL-grown culture is shown in FIG. 3A.Although HSL and HS elute at similar times, both compounds were resolvedby extracting the M+1 molecular ions 102 and 120, respectively, from theraw chromatogram (a standard MS practice).

P. aeruginosa PAO1 growth and metabolism of 3OC12HSL as the sole energysource in “MES 5.5” medium is shown in FIG. 4. By early stationaryphase, all of the white, nearly insoluble 3OC12HSL-substrate wasconsumed such that concentrations of less than 125 nM remained. HSLaccumulated throughout the growth phase and reached a maximum of ca. 500μM just before the onset of stationary phase, after which it decreasedto less than 80 μM by 100 hours into stationary phase. Concomitant withthe disappearance of HSL, the amino acid homoserine accumulated and thendecreased to concentrations below 80 μM by 100 hours into stationaryphase (FIG. 4). Since the culture pH was well-controlled at pH 5.5, andsince the half life decay of HSL into homoserine at this pH is on theorder of weeks, an enzymatic HSL lactonase, not abiotic alkalinehydrolysis, is most likely responsible for the evolution of homoserine.P. aeruginosa did not grow using either HSL or homoserine as a soleenergy source in “MES 5.5” media. When provided with long chain AHLs assole sources of carbon and nitrogen, strain PAO1 grew at rates abouttwice as slowly as cultures utilizing AHL plus ammonium (not shown).Cells did not use either homoserine or HSL as sole sources of energy ornitrogen.

Other Analyses.

Strain PAI-A was examined for several traits exhibited by P. aeruginosa,which was used as a positive control. Fluorescent pigment production wasexamined using Wood lamp illumination of colonies grown on LB agar.Pyocyanin production was examined in glycerol-alanine medium (11).Production of acyl-HSLs in both LB and defined media was examined usingpreviously described radioassay methods (33). A Beckman System Gold HPLCrunning a methanol gradient was used in the chromatographic analysis ofethyl acetate extracts as previously described (34). Radioactivity wasmonitored via on-line, solid scintillation counting using an in-lineHPLC β-particle detector (IN/US Model 3, Tampa, Fla.). Microscopicexaminations were performed using a Zeiss Stemmi 2000 stereomicroscope(low-magnification), and a Zeiss Axioplan research microscope (highermagnification, phase-contrast and dark-field). Nitrate-dependentanaerobic growth was tested using both MOPS-buffered defined media or LBamended to contain 10 mM potassium nitrate dispensed under a 100% N₂headspace in Bellco (Vineland, N.J.) 18-mm butyl, serum-stoppered“Balch” tubes.

Analysis of Cell-Free Culture and Reaction Fluids.

For the initial characterization of the intermediates in AHLdegradation, a TLC method was used (16). For a refined analysis, aliquid chromatography-atmospheric pressure chemical ionization massspectrometry (LC/APCI-MS) technique was developed to monitor andquantify the disappearance of AHL and appearance of a number of AHLdegradation products. For this analysis, 50 μl samples of culture fluidswere taken in triplicate from AHL-grown cultures and were centrifuged at15,800× g for 10 minutes. The cell-free culture fluids were stored at−20° C. until all samples had been collected for analysis. For LC/MSanalysis, samples were mixed 1:1 with acetic acid-acidified methanol (1%vol/vol). Dilutions were made using “MES 5.5” medium. A C18 ultraaqueous reverse phase column (5 μm bead size, 50 mm×3.2 mm; Restek No.317553) was employed. The initial mobile phase was 50:50:1methanol:water:acetic acid running at 0.5 ml·min⁻¹ isocratically overthe first minute after injection and increased (via a linear gradient)to 80:20:1 methanol:water:acetic acid over the following 2 minutes (FIG.3). Using this method, a diversity of AHLs, their correspondingacyl-homoserines, HSL, and homoserine in samples could be quantifiedfrom cultures growing in the “MES 5.5” defined medium. These analyseswere performed at Caltech's Environmental Analysis Center using aHewlett Packard 1100 Series LC/APCI-MS mass spectrometer.

Standards over a range of concentrations (125 nM to 1 mM) were preparedusing either water or “MES 5.5” basal media depending on the origin ofthe sample, and were diluted 1 to 1 with acetic acid acidified methanol(1% vol/vol). For a 20 μl sample injection, the detection limits forstandards prepared in “MES 5.5” media were: 2.5 pmoles for 3OC12HSL andits corresponding acyl-homoserine, 10 pmoles for C10HSL and itscorresponding acyl-homoserine, and 100 pmoles for HSL. The limit ofdetection for standards prepared in water was lower than for thoseprepared in medium, but the former but were only used to quantify AHLsrecovered from evaporated ethyl acetate extracts. Ethyl acetateextraction did not recover HSL or homoserine. The accurate quantizationof homoserine in standards and samples was complicated by its partiallactonization into HSL, a chemical reaction that occurred afterinjection into the LC/APCI-MS instrument. Thus, while homoserine plusHSL pool sizes could accurately be quantified, homoserine itself wasdetermined with less precision using the LC/APCI-MS method and wasusually a slight underestimate.

Nucleotide Sequence Analysis of the SSU rDNA Strain PAI-A.

The nucleotide sequence of a PCR-amplified fragment of the 16S rDNA ofstrain PAI-A was determined and analyzed using previously describedprocedures (20, 21). Sequence reads were assembled and edited usingSequencher (Genecodes, Ann Arbor). Multiple sequence alignments,translations, and phylogenetic analyses were performed using the LinuxARB freeware package (www.arb-home.de/). Phylograms were constructed viaPuzzle-Map 5.0 maximum likelihood analyses (36). Tree layout wasperformed using Treeview 1.6.6 for Windows (30). The 1401 base pairsequence for strain PAI-A has been submitted to GenBank (AY288072). TheGenBank accession numbers for the other sequences presented in FIG. 2are as follows: Pseudomonas strain CRE 11, U37338 (28); P. aeruginosaPAO1, AE004949 (38); Pseudomonas strain BD1-3, AB015516; P.anquilliseptica, X99540 (5); P. balearica U26418 (3); P. resinovorans,AB021373 (1); P. oleovorans, D84018 (2); P. citronellolis, Z76659 (26);Pseudomonas strain 273, AF039488 (44); Pseudomonas strain B13, AJ272544(24); P. nitroreducens, D84021 (2); and Pseudomonas strain CRE 12,U37339 (28).

Phylogenetic Analysis of Strain PAI-A.

A nearly complete sequence of the SSU rDNA was obtained. Web-basedsimilarity searches against rDNA in the RDP-II and GenBank databasessuggested that strain PAI-A was most closely related to P. aeruginosaand several other pseudomonads. The small subunit (SSU) rDNA shared98.4% and 98.1% sequence identity with P. aeruginosa PA01 and P.resinovorans, respectively. By any of the distance, parsimony andmaximum likelihood methods employed (FIG. 2), the SSU rDNA from str.PAI-A clustered most closely with those from P. aeruginosa and its closerelatives.

Properties of Pseudomonas PAI-A. Strain PAI-A grew aerobically in bothdefined media and LB at 30° and 37°, but not at 42° C. Cultures doubledevery 35 minutes in defined medium with succinate as the sole carbonsource at 37° C. The isolate grew on a number of tested substrates atboth pH 7.2 and pH 5.5; however, cultures did not grow in AHL-containingmedia at the latter pH. Cells did not grow anaerobically in eithersuccinate-defined or LB media amended with nitrate. Exponentiallygrowing cells sampled from AHL-containing media were vigorously motilerods, 2.5×0.8 pm in dimension. The isolate formed creamy-white colonieswith spreading edges. After several days, colonies become smooth,non-sticky, leathery, and extremely recalcitrant to disruption with aninoculating loop.

Strain PAI-A did not produce colored or fluorescent pigments in or on LBor glycerol-alanine (pyocyanin-production) media. Cultures did have anyaroma of note. Cells did not grow in media containing 30 μgnalidixate·ml⁻¹. To examine Pseudomonas PAI-A for the production of AHLquorum signals, cultures grown in both defined and LB media wereincubated with ¹⁴C-carboxyl methionine. Since no radioactive peaks wereevident after chromatography of the ethyl acetate extract fraction, thisisolate does not appear to accumulate AHLs under the conditionsexamined.

Example 2 Characterization of Pseudomonas AHL Acylase Activity

Cloning and Expression of pvdQ (PA2385) Encoding a Putative P.aeruginosa AHL Acylase.

Genomic DNA was isolated from P. aeruginosa PAO1 using the DNeasy™tissue kit (QIAGEN) and used as a template for PCR. The deduced codingregion for PvdQ (Gene PA2385; www.pseudomonas.com) was amplified fromthe genomic DNA using the following primers:5′-AGGCCAAGCTTATGGGGGATGCGTACCGTACTG-3′ (SEQ ID NO:6) and5′-GTTATATAGCGGCCGCTAGGCATTGCTTATCATTCG-3′ (SEQ ID NO:7; bold printindicates HindIII and NotI restriction sites, respectively), cloned intothe appropriately digested expression vector, pPROTet.E133 (Clontech),and transformed into E. coli BL21 PRO. Recombinant AHL acylase activitywas examined as follows. After growth in LB medium containingspectinomycin (50 μg·ml⁻¹) and chloramphenicol (34 μg·ml⁻¹), and aftergene induction by the addition of anhydrotetracycline (aTc, 100 ng·ml⁻¹)at 18° C., cells were pelleted and resuspended to a final opticaldensity of 1.2 in MOPS buffered media (pH 7.2) containing 10 μM3OC12HSL. Recombinant cells that had not been induced with aTc were usedas a negative control. Reaction mixtures were incubated at 18° C.; 150μl samples were removed at 0, 15, 30, and 60 min and analyzed for AHLdisappearance and product appearance via LC/APCI-MS (see above).

Constitutive Overexpression of PvdQ in P. Aeruginosa.

For the constitutive expression of PvdQ in strain PAO1, the codingsequence was PCR-amplified with the following primers:5′-AAGAGGACATATGGGGGATGCGTACCGTACTG-3′ (SEQ ID NO:8) and5′-CTAAAGCTTGGCTGTGGGCCGCCTCTATGG-3′ (SEQ ID NO:9; bold print indicatesNdeI and HindIII restriction sites, respectively). The PCR product wascloned into the E. coli-Pseudomonas shuttle expression vector pUCP-Ndedigested with NdeI and HindIII (4). The resulting construct, pPvdQ-Nde,was transformed into P. aeruginosa PAO1 via electroporation. Since therepression of gene expression from this vector requires LacI, and sincewild-type P. aeruginosa PAO1 does not encode this repressor, theprobable acylase was expected to be constitutively expressed, aprediction borne out after the examination of total cell proteins viaPAGE.

Analysis and Expression of P. Aeruginosa PvdQ, which Encodes a CandidateAHL Acylase.

The P. aeruginosa gene PA2385 (recently named PvdQ (18)), which wasidentified as a close homologue to a gene encoding an HSL-releasing AHLacylase from Ralstonia XJ 12B (23), was examined to determine whether itencoded a protein with AHL acylase activity and conferred theAHL-dependent growth of P. aeruginosa. The coding region of this genewas amplified from the genomic DNA, cloned into an expression vector,and expressed in E. coli. The polypeptide encoded by the gene waspredicted to be postranslationally cleaved into two distinct subunits.PAGE analysis of the total proteins fraction from E. coli cellsexpressing recombinant PvdQ revealed small amounts of the two expectedsubunits. The majority of the recombinant protein was recovered as theunprocessed 80 kDa propeptide. This observation is similar to that notedby Zhang and co-workers for recombinant AiiD, the Ralstonia AHL acylase(23).

Resting cell suspensions expressing PvdQ were incubated with 10 μM3OC12HSL, which is a concentration relevant to the quorum sensingphysiology of P. aeruginosa. AHL disappearance and the appearance of HSLand 3OC12-homoserine were evaluated using the LC/APCI-MS analysis ofcleared reaction fluids (FIG. 5). Within an hour, the AHL disappearedconcurrent with the accumulation of stoichiometric amounts of HSL asproduct. No 3OC12-homoserine accumulation was observed. Cell-free anduninduced cell controls did not catalyze HSL release or the degradationof the AHL over the same time period. In a pattern similar to the AHLutilization data (Table 1), cells of E. coli expressing the recombinantacylase catalyzed the HSL-releasing degradation of C14HSL, C12HSL,C10HSL. and C8HSL, but not 3OC6HSL or C6HSL. The effects of theconstitutive expression of PvdQ in P. aeruginosa PAO1 were alsoexamined. In comparison to wild-type, which accumulated 3OC12HSL toconcentrations in excess of 6 μM during growth in LB at 30° C. (FIG. 6),cultures expressing the acylase did not accumulate any of this quorumsignal above the threshold of detection.

A PvdQ deletion-replacement mutant and the strain QSC112a, which carriesa Tn5-insertion into PvdQ, were also examined for growth in definedmedia with 3OC12HSL as sole energy source. Both mutants grew with thesame growth rates and yields as wild-type (data not presented).Accumulations of endogenously produced 3OC12HSL by LB-grown cultures ofthe PvdQ-deletion mutant were identical to that of the wild-type grownunder the same conditions. Evidently, although PvdQ encodes an enzymewith an HSL releasing AHL acylase activity specific towards long-chainAHLs, another enzyme must be a significant contributor to the growthphenotype on AHL.

Each of the following publications is incorporated herein by reference.

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Although the invention has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

What is claimed is:
 1. A method of identifying an agent that modulatesγ-proteobacterium long chain acyl homoserine lactone (AHL) acylaseactivity, comprising: a) contacting at least one sample comprisingPA1032 AHL acylase as set forth in SEQ ID NO: 5 and a long chain AHLwith at least one test agent, under conditions suitable for AHL acylaseactivity, wherein the PA1032 AHL acylase is truncated from about 1 to 6amino acids at the NH terminus and comprises an alternate methionineinitiator residue; and b) measuring AHL acylase activity in the presenceof the test agent; wherein a change in AHL acylase activity as comparedto the AHL acylase activity in the absence of the test agent identifiesthe test agent as an agent that modulates the γ-proteobacterium longchain AHL acylase activity.
 2. The method of claim 1, wherein the longchain AHL comprises N-3-octanoyl-DL-homoserine lactone (C8HSL);N-3-decanoyl-DL-homoserine lactone (C10HSL);N-3-dodecanoyl-DL-homoserine lactone (C12HSL);N-3-oxododecanoyl-L-homoserine lactone (30C12HSL); orN-3-tetradecanoyl-DL-homoserine lactone (C14HSL).
 3. The method of claim1, wherein the γ-proteobacterium comprises a Pseudomonas species.
 4. Themethod of claim 2, wherein the Pseudomonas species comprises Pseudomonasaeruginosa.
 5. The method of claim 1, wherein the sample furthercomprises a short chain AHL, and wherein the method further comprisesdetecting no change in the amount of short chain AHL in the presence ofthe test agent as compared to the absence of the test agent.
 6. Themethod of claim 5, wherein the short chain AHL comprisesN-3-butanoyl-DL-homoserine lactone (C4HSL); N-3-hexanoyl-L-homoserinelactone (C6HSL); N-3-oxohexanoyl-L-homoserine lactone (30C6HSL); orN-3-heptanoyl-DL-homoserine lactone (C7HSL).
 7. The method of claim 1,wherein the modulating comprises increasing the AHL acylase activity. 8.The method of claim 1, wherein the sample comprises a cell free sample.9. The method of claim 1, wherein the AHL acylase comprises purified AHLacylase or an extract comprising a γ-proteobacterium.
 10. The method ofclaim 3, wherein the Pseudomonas species comprises a knock-out of thewild type AHL acylase gene.
 11. The method of claim 1, wherein thesample is a prokaryotic cell transformed with a vector encoding theamino acid sequence as set forth in SEQ ID NO:5.
 12. The method of claim1, wherein detecting a change in AHL acylase activity comprisesmeasuring AHL levels in the sample.
 13. The method of claim 1, whereincontacting the at least one test agent comprises contacting a pluralityof different test agents each with their own sample.
 14. The method ofclaim 13, wherein the different test agents comprise a library of testagents.
 15. The method of claim 14, wherein the library of test agentscomprises a combinatorial library of test agents.
 16. The method ofclaim 15, wherein the combinatorial library comprises a random library,a biased library, or a variegated library of test agents.
 17. The methodof claim 1, wherein the test agent modulates the activity of PA1032 AHLacylase by modulating expression of a nucleic acid sequence encoding forPA1032 AHL acylase encoded by nucleotides 19 to 2544 of SEQ ID NO: 3,wherein nucleotides 19 to 21 have an alternative initiator methionineresidue.
 18. The method of claim 17, wherein the agent increases AHLacylase gene expression, thereby increasing AHL acylase activity.
 19. Amethod of identifying an agent that modulates γ-proteobacterium longchain acyl homoserine lactone (AHL) acylase activity, comprising: a)contacting at least one sample comprising PA1032 AHL acylase as setforth in SEQ ID NO: 4 or SEQ ID NO: 5 and a long chain AHL with at leastone test agent, under conditions suitable for AHL acylase activity; andb) measuring AHL acylase mRNA levels in the presence of the test agent;wherein a change in AHL acylase mRNA levels as compared to the AHLacylase mRNA levels in the absence of the test agent identifies the testagent as an agent that modulates the γ-proteobacterium long chain AHLacylase activity.
 20. The method of claim 19, wherein the mRNA levelsare measured using an assay selected from the group consisting ofmicroarray analysis, Northern blot analysis, and promoter fusionanalysis.